Purification of lineage-specific cells and uses therefor

ABSTRACT

A method for developing a population of substantially lineage-specific cells and their use inter alia in tissue replacement therapy, tissue augmentation therapy, diagnostic applications, for the identification of growth factors and other autocrine factors. Specifically, substantially homogeneous populations of mammalian cells of the astrocyte lineage are provided and selected on the basis of differential marker expression.

FIELD OF THE INVENTION

The present invention relates generally to a method for developing apopulation of substantially lineage-specific cells and their use interalia in tissue replacement therapy and tissue augmentation therapy andin diagnostic applications and for the identification of growth factorsand other autocrine factors such as expansion, proliferation anddifferentiation factors. More particularly, the present inventionprovides mammalian cells of the astrocytic lineage cells obtainable frommammalian brains such as from embryo brain tissue or parts thereof suchas the retina and selected on the basis of differential markerexpression. The ability to selectively enrich or obtain or otherwisegenerate a pure homogeneous population and preferably a pure populationof astrocyte precursor cells and immature perinatal astrocytes permitstissue replacement and augmentation therapy of the brain resulting froma degenerative and in particular a neurodegenerative or other diseaseconditions or trauma. The cells may be derived from the subject to betreated (i.e. autologous transplantation/augmentation therapy) or may bederived from suitably histocompatibility matched individuals(heterologous or non-autologous transplantation/augmentation therapy).The identification of markers specific for certain developmental stagesof astrocytic lineage along its maturation pathway permits thedevelopment of assays to distinguish between the developmental stagesand this is useful in the development of diagnostic and therapeutictools. Factors identified and obtainable from cultured astrocyteprecursor cells or immature perinatal astrocytes are useful infacilitating tissue replacement and augmentation therapy and in inducingtissue repair and regeneration. These factors may also be administereddirectly into the brain or other suitable location to facilitatedevelopment of lineage-specific cells.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Multipotent stem cells are undifferentiated cells which are capable ofdifferentiation and proliferation into multiple cell lineages and typesand have the ability of self-renewal.

During development of the central nervous system (CNS), multipotent stemcells which have the capacity of generating many types of neurons andglia, give rise to precursor cells that are progressively morerestricted in differentiation potential. Although substantial progresshas been made in understanding the development of oligodendrocytes andSchwann cells, much less is known about astrocyte development.Furthermore, whereas several types of multipotent stem cells andlineage-restricted precursor cells have been characterized and appliedclinically in recent years, the lack of knowledge of the sequence ofevents that underlies astrocyte development has limited the success ofsuch applications.

It has been shown that a glial-restricted precursor (GRP) cell isolatedfrom the spinal cord of mice on embryonic day (E) 13.5 is capable ofgiving rise to oligodendrocytes and both type-1 and type-2 astrocytes inculture. However, the intermediate stages of differentiation between theGRP cell and mature, differentiated astrocytes present in the adult CNSare not well characterized. There is a need, therefore, to increase theunderstanding of the developmental biology of cells of the astrocyticlineage.

Early studies provided evidence for the existence of astrocyte precursorcells (APCs) that give rise only to astrocytes (Raff et al., Devel.Biol. 106: 53-60, 1984; Fok-Seang, J. and Miller, R. H., J. Neurosci.12: 2751-2764, 1992; Davis, A. A. and Temple, S., Nature 372: 263-266,1994; Levison, S. W. and Goldman, J. E., J. Neurosci. Res. 48: 83-94,1997; Mi. J. and Barres. B. A., J. Neurosci. 19: 1049-1061, 1999). Suchcells present in cultures of neonatal rat spinal cord were characterizedas highly migratory as well as positive for the A2B5 antigen andvimentin and negative for glial fibrillary acidic protein (GFAP) andgalactocerebroside (Fok-Seang and Miller, 1992, supra). Such cells arealso present in neonatal rat optic nerve and were characterized aspositive for Pax2, A2B5, C5, Ran-2 and Vimentin and negative for GFAP,S100β, and weakly positive for nestin (Ni and Barres, 1999, supra).

Little is known of the characteristics of APCs in vivo. Until the adventof the present invention, the existence of an APC that gives rise onlyto astrocytes in the developing human CNS and adult CNS has notpreviously been demonstrated. Furthermore, immunohistochemical and insitu hybridization analyses have shown that, in the mouse cerebellum,Pax2 (Mi and Barres, 1999, supra) is not expressed by cells of theastrocytic or oligodendrocytic lineages, but is rather localized toγ-aminobutyric acid-containing interneurons and deep cerebellar nuclei(Maricich, S. M. and Herrup, K., J Neurobiol. 41: 281-294, 1999). Thereis an apparent discrepancy, therefore, in Pax2 gene expression datafollowing in vitro and in vivo studies of CNS development. Furthermore,until the advent of the present invention, there was a lack of in vivostudies of APC differentiation in human fetal tissue.

The Pax2 gene is a member of the Pax gene family which encodestranscription factors, all of which are DNA-binding proteins thatcontain a paired-box domain. Each member of the Pax family is expressedin a spatially and temporally restricted manner, suggesting that theseproteins contribute to the control of tissue morphogenesis and patternformation. Pax2 is implicated in organogenesis of the kidney, eye, ear,and the CNS. Heterozygous mutations in the Pax2 gene result in failureof the optic groove to form in the mouse optic nerve (Otteson et al.,Devel. Biol. 193: 209-224, 1998) and are associated in humans and micewith optic nerve coloboma (Sanyanusin et al., Nature Genetics 9:358-364, 1995; Favor et al., Proc. Natl. Acad. Sci. USA 93: 13870-13875,1996), a condition characterized by enlargement and blurring of themargin of the optic disk. Homozygous mutations in the Pax2 gene resultin retinal coloboma as a consequence of failure of the retinal fissureto close (Torres et al., Development 122: 3381-3391, 1996).

Pax2 expression during ocular development has been studied in mice,rats, and humans. Transcripts of the Pax2 gene are first apparent in thedeveloping mouse eye on embryonic day (E) 9 and are initially restrictedto the ventral optic cup and stalk (Nornes et al., Development 109:797-809, 1990; Otteson et al., 1998, supra). By E16.5, these transcriptshave disappeared from the ventral retina and are present in a ring ofcells around the optic nerve head (ONH) and in the parenchyma of theoptic nerve. At E18, Pax2 mRNA is apparent on the vitreal surface of theposterior retina, consistent with the timing and topography of astrocytemigration into the mouse retina. Pax2 mRNA was not detected in theretina, optic disk, or optic nerve of adult mice (Otteson et al., 1998,supra). In the rat optic nerve, Pax2 expression is already widespread atE17 (Mi and Barres, 1999, supra). Although the pattern of Pax2expression during development of the rat optic nerve is consistent withthe observations in the mouse, Pax2 expression persists at a low levelin the adult rat nerve. Limited observations in humans have shown that,between 6 and 8 weeks of gestation (WG), Pax2 is expressed in the regionof the optic disk and nerve (Terzic et al., Int. J. Dev. Biol. 42:701-707, 1998). Despite the documentation of Pax2 expression duringearly embryonic development of the optic nerve and eyecup until theadvent of the present invention, the relation between Pax2 expressionand differentiation of the astrocytic lineage during the later stages ofretinal development has been unknown.

Various studies have examined the development of GFAP⁺ astrocytes invivo. In the human retina, astrocytes with two distinct morphologies andlocations have been described: those with parallel processes closelyassociated with nerve fiber bundles (NFBs), and star-shaped astrocytespresent in the ganglion cell layer (GCL) that often ensheath bloodvessels (Wolter, J., Am. J: Ophthal 40: 88-99, 1955; Ogden, T. E.,Invest. Ophthalmol. Vis. Cii. 17: 499-510, 1978; Ramirez et al., Vis.Res. 34: 1935-1946, 1994; Trevino et al., Vis. Res. 37: 1707-1711, 1997;Provis et al., Exp. Eye Res. 65: 555-568, 1997; Hughes et al., Invest.Ophthalmol. Vis. Sci. 41: 1217-1228, 2001). Astrocytes first appear inthe monkey retina around the optic disk and spread peripherally,reaching the edge of the retina before birth (Gariano et al, Invest.Ophthalmol. Vis. Sci. 37: 2367-2375, 1996); vimentin and GFAPimmunohistochemical analysis of retinal sections revealed that immaturespindle-shaped astrocytes precede the developing vasculature. Astrocytesalso precede the formation of blood vessels by a small margin in thehuman (Chan-Ling et al., Proc. Aust. Neurosci. Soc. 7: 48, 1996; Proviset al., 1997, supra; Hughes et al., 2001, supra), cat (Ling, T. andStone, J., Dev. Brain Res. 44: 73-85, 1988; Chan-Ling, T. and Stone, J.,J. Comp. Neurol. 303: 387-399, 1991), and rat (Ling et al., J. Comp.Neurol. 286: 345-352, 1989) retina and are thought to secrete vascularendothelial growth factor (VEGF), which mediates hypoxia-inducedangiogenesis (Chan-Ling et al., Invest. Ophthalmol. Vis. Sci. 36:1201-1214, 1995; Stone et al., J. Neurosci. 15: 4738-4747, 1995; Hugheset al, 2000, supra).

In work leading to the present invention, the present inventorinvestigated the relationship between Pax2 expression and cells of theastrocytic lineage in the human retina and the optic nerve head (ONH),and characterized the time course of appearance and topography of spreadof APCs and perinatal astrocytes in the human retina.

The subject inventor identified positive and negative markers which werespecific for particular developmental stages during maturation ofastrocytic lineage. In accordance with the present invention, thesemarkers in combination with other in vitro markers, are used toselectively enrich or generate populations of APCs or immature perinatalastrocytes (IPAs) or other astrocyte cells such as mature perinatalastrocytes. Furthermore, in accordance with the present invention, APCsare identified in the adult human brain. The ability to generate suchpopulations permits their developmental expansion for use in tissuereplacement and augmentation therapy and to identity factors involved intheir proliferation and differentiation. The identification of immatureAPCs in the adult human brain is particularly significant in terms of asource of cells for autologous therapy. Furthermore, the presentinvention encompasses antagonists and agonists of these factors as wellas naturally occurring molecules which inhibit proliferation,differentiation and/or growth of these cells.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The present invention identifies astrocyte cell markers which arecapable of distinguishing between developmental stages. From multipotentstem cells, lineage-specific APCs are formed expressing Pax2 andvimentin but not either glial fibrillary acid protein (GFAP) or S-100.The next developmental stage is the formation of IPAs which express allfour of the above markers. Mature perinatal astrocytes (MPAs) lose theability to express vimentin and then adult astrocytes further lose Pax2expression as a function of physiologic aging. The ability toselectively enrich cultures of cells for APCs or IPAs permits their usein tissue replacement and augmentation therapy. Importantly, APCs havebeen identified in accordance with the present invention in adult brainas well as the retina and, hence, this aspect represents a source ofAPCs for autologous therapy as well as for heterologous therapy.Furthermore, homogeneous populations of APCs or IPAs can be used toisolate particular growth or autocrine factors for use in conjunctivetherapy to tissue replacement and augmentation therapy or to inducerepair or regeneration of endogenous tissue. The markers further permitmixed populations of astrocytes in various stages of development to beidentified and this has diagnostic and therapeutic applications.

Accordingly, one aspect of the present invention contemplates a methodfor generating a substantially homogeneous population oflineage-specific cells from tissue of the central nervous system (CNS)of mammalian animals, said method comprising subjecting said CNS tissueto tissue disruptive means comprising the lineage-specific cells to beisolated, subjecting the mixed population to cell separationdiscrimination means to generate a substantially homogeneous populationof lineage-specific cells.

Preferably, the lineage-specific cells are APCs or IPAs from tissue ofthe CNS such as brain including retina tissue.

The present invention contemplates, therefore, a method of generating asubstantially homogeneous population of APCs from tissue of the CNS suchas from brain or parts thereof including the retina or parts thereof,said method comprising subjecting said CNS tissue to tissue disruptivemeans to produce a population comprising APCs amongst other cells andsubjecting said population of cells to a cell sorting methodologyincluding such as subjecting cells to positive selection using surfacemarkers GD3, A2B5, C3B2, FGFR3 and/or PDGFRα or a combination thereof,then subjecting the positively selective cells to negative selectionusing GlC, 01, 04, anti-Mog and/or NG2 or a combination thereof. Theidentity of the purified population of cells is confirmed using Pax2,vimentin, GFAP and S-100 immunohistochemically.

Having obtained the APCs, a substantially homogenous population of IPAsor a mixed population of IPAs and APCs are included along the maturepathway is induced using one or a combination of inter alia CNTF, LIF,BMP (e.g. BMP4), TGFβ, cAMP and EGF.

In a most preferred embodiment, the cells are purified as follows. Apopulation of cells is selected and single cell suspensions prepared.Using negative selection such as N-CAM (also known as PSA-N-CAM neuralcells are removed from their cell population. Glial cells are positivelyselected using markers such as A2B5, GD3, 3CB2, FGFR3, PDGFRα or acombination thereof. The cells are then cultured in a serum free mediumsuch as DMEM/F-12 supplemented with growth factors such as bFGF andchick embryo extract. In the resulting population, oligodendrocytes areremoved using markers such as GlC, 01, 04, Gal-C, anti-MOG and NG2. Theresulting population is induced to differentiate along the maturationpathway using growth factors such as CNTF, LIF, BMP such as BMP4, cAMP,TGFβ and EGF. The cells can then be characterized immunohistochemicallybased on the markers presented in Table 1.

The present invention provides, therefore, a substantially homogeneouspopulation of mammalian lineage-specific cells from the CNS. Thepreferred mammalian lineage-specific cells are APCs or IPAs.

Another aspect of the present invention contemplates a method of cellreplacement therapy in a mammalian animal, said method comprisinggenerating a substantially homogeneous population of lineage-specificcells and introducing same into an organ or tissue requiring cells to bereplaced or to another location from where the cells can migrate to anorgan or tissue requiring cells wherein the introduced cells are subjectto expansion or proliferation in vitro and/or in vivo by one or moregrowth factors. Generally, the lineage-specific cells are APCs or IPAs.The APCs or IPAs or tissues derived therefrom may be from the subjectbeing treated (i.e. autologous therapy) or from a suitablyhistocompatibility matched subject (i.e. heterologous therapy).Autologous therapy is preferred.

Yet another aspect of the present invention provides a composition ofastrocyte precursor cells such as APCs or IPAs in substantiallyhomogeneous form, said composition optionally further comprising one ormore pharmaceutical acceptable carriers and/or diluents.

Still another aspect of the present invention contemplates a growth orautocrine factor obtainable from conditioned medium of an in vitro cellculture of astrocyte precursors such as APCs or IPAs. The growth orautocrine factor may be used in vitro to expand a population oflineage-specific cells or may be administered directly to the brain tofacilitate or promote development of replacement cells.

In a related embodiment, the present invention proposes the use ofmicroarray technology and differential expression arrays to determinecell surface markers including differentially expressed cell surfacemarkers at different stages of astrocyte cell development. Such studiesassist in the identification of growth factor receptors for use inselecting growth and autocrine factors to promote proliferation and/ordifferentiation of particular astrocyte cells.

A further aspect of the present invention contemplates the use of thepurified astrocytes and in particular APCs and IPAs as gene therapycarriers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photographic representation showing: (A) Cryostat section ofa human retina at 24 to 26 weeks gestation labeled with both anti-Pax2(red) and anti-GFAP (green). Pax2⁺, GFAP⁻ APCs (arrow) and Pax2⁺, GFAP⁺perinatal astrocytes (arrowhead) were detected only within the NFL andGCL. Pax2 expression was apparent only in the cell somas.Autofluorescent granules were observed in the RPE. (B and C) Retinalwhole-mounts triple-labeled with anti-Pax2 (red), anti-vimentin (green),and anti-GFAP (blue) at 12 weeks gestation. (B) At the posterior pole,APCs were Pax2⁺, GFAP⁻, and vimentin⁺ (arrow), whereas perinatalastrocytes were Pax2⁺, GFAP⁺, and vimentin⁺ (arrowheads). (C) At theleading edge of Pax2 expression, APCs were Pax2⁺, GFAP⁻, and vimentin⁺(arrows). (D and E) Retinal whole-mount at 32 weeks gestationdouble-labeled for Pax2 (red) and GFAP (green). (D) Toward the retinalperiphery, Pax2⁺, GFAP⁺ perinatal astrocytes with bipolar processes werelocated in superficial layers. (E) Vascular layer of the same region ofthe retina as that shown in (D), revealing Pax2⁺, GFAP⁺ perinatalastrocytes closely associated with blood vessels. (F) Posterior regionof a retinal whole-mount at 32 weeks gestation with Pax2 (red) andvimentin (red) labeling. Vimentin filaments (arrows) were restricted toonly a few Pax2⁺ cells. (G) Retinal whole-mount at 17 weeks gestationlabeled with anti-GFAP (brown). Perinatal astocytes were closelyassociated with NFBs. (H) Retinal whole-mount at 17 weeks gestatationdouble-labeled with anti-CD34 (pink) and anti-GFAP (brown). At theleading edge of vessel formation, perinatal astrocytes preceded theformation of patent blood vessels by a small margin. Color versions ofthis photograph are available from the patentee.

FIG. 2 is a photographic representation showing: (A through F) Cryostatsection of a retina at 24 to 26 weeks gestation labeled with bothanti-Pax2 (red) and anti-GFAP (green). Posterior (A and B), equatorial(C and D), and peripheral (E and F) regions are shown. APCs (arrows) andperinatal astrocytes (arrowheads) are indicated. (A and B) Pax2⁺, GFAP⁻APCs were observed in the superficial layer of the NFL. (E) Only Pax2⁺,GFAP⁻ APCs were apparent peripherally. (F) At the retinal edge, neitherPax2⁺, GFAP⁻ APCs nor Pax2⁺, GFAP⁺ perinatal astrocytes were detected.(G) Photographic montage of the ONH region of a retina at 8 weeksgestation labeled with both anti-Pax2 (red) and anti-GFAP (green). (H) Atracing of the montage in (G) showing the location of individual Pax2⁺,GFAP⁻ APCs and Pax2⁺, GFAP⁺ perinatal astrocytes. Two clusters of APCsand perinatal astrocytes are present at the ventricular zone surroundingthe ONH. (and J) Adjacent sections of a retina at 24 to 26 weeksgestation double-labeled with anti-Pax2 (red) and anti-GFAP (green) (I),or stained with toluidine blue (J), showing the ventricular zone at highmagnification. (I) APCs (arrow) and perinatal astrocytes (arrowhead)were observed in the ventricular region. Autofluorescent granules wereapparent in the RPE. Color versions of this photograph are availablefrom the patentee.

FIG. 3 is a photographic representation showing: (A through C) Retinalwhole-mounts positioned with the RPE uppermost and showing triplelabeling of the ONH region at 14 to 16 weeks gestation with anti-Pax2(red), anti-vimentin (green), and anti-GFAP (blue). (A) The ONH islocated at the top left of the image. At the ventricular zone, the ONHis surrounded by Pax2⁺, GFAP⁺, vimentin⁺ perinatal astrocytes(arrowhead) and an outer layer of Pax2⁺, GFAP⁻, vimentin⁺ APCs (arrow).(13) Higher magnification of the region in (A), showing the layers ofAPCs and perinatal astrocytes. (C) Higher magnification of the region in(A), showing the outer layer of Pax2⁺, GFAP⁻, vimentin⁺ APCs. (D) Theventricular zone of a retinal whole-mount at 32 weeks gestationdouble-labeled with anti-Pax2 (red) and anti-GFAP (green). The ONH islocated beyond the top left corner of the image. It is still surroundedby Pax2⁺, GFAP⁺ perinatal astrocytes and an outer layer of Pax2⁺, GFAP⁻APCs. (E through G) Schematic representations of the distributions ofPax2⁺, GFAP⁻, vimentin⁺ APCs and Pax2⁺, GFAP⁺, vimentin⁺ perinatalastrocytes around the ONH at 14 to 16 weeks gestation (E), 24 to 26weeks gestation (F), and 32 weeks gestation (G). Color versions of thisphotograph are available from the patentee.

FIG. 4 is a graphical representation of topographic maps of the outerlimits of APCs and perinatal astrocytes in the human retina at 12, 16,18, 21, 22 to 23, 26, 28, and 32 weeks gestation as well as thedistributions of perinatal astrocytes and adult astrocytes in the agedadult human retina. In the fetal retinas, red dots indicate the area ofAPCs and purple dots show the area of perinatal astrocytes. With theexception of a rim at the leading edge of astrocyte migration, whereonly APCs are found, both APCs and perinatal astrocytes are presentinterspersed over the central region of the retina during fetaldevelopment. The macular region does not contain either of these celltypes. The distributions of Pax2⁺, GFAP⁺, vimentin mature perinatalastrocytes (purple dots) and Pax2⁻, GFAP⁺, vimentin⁻ adult astrocytes(blue dots) in the aged adult human retina are shown in the bottom twomaps. Scale bars, 1 mm (12 weeks gestation) or 10 mm (all other ages).Color versions of this photograph are available from the patentee.

FIG. 5 is a photographic representation showing (A and B) Retinalwhole-mount at 16 weeks gestation triple-labeled with anti-Pax2 (red),anti-vimentin (green), and anti-GFAP (blue). (A) At the leading edge ofPax2 labeling, APCs were Pax2⁺, vimentin⁺, and GFAP⁻. (B) Highermagnification of the same region of the retina shown in (A). (C and D)The foveal and raphe regions of a retinal whole-mount at 18 weeksgestation double-labeled with anti-Pax2 (red) and anti-GFAP (green). (C)Arrowheads indicate the border of the presumptive fovea. Pax2⁺, GFAP⁻APCs and Pax2⁺, GFAP⁺ perinatal astrocytes were not detected in thepresumptive foveal zone. Small arrow points towards representative APCsand large arrow points towards a perinatal immature astrocyte in theperifoveal region. (D) Higher magnification of the boxed region in (C),showing the border zone. (E and F) The foveal and raphe regions ofretinal whole-mounts at 18 weeks gestation labeled with anti-GFAP(brown). Perinatal astrocytes follow the path of NFBs in the rapheregion. (G) Adult retinal whole-mount triple-labeled with anti-Pax2(red), anti-vimentin (red) and anti-GFAP (green) and showing thepresence of Pax2⁺, GFAP⁺, vimentin⁻ mature perinatal astrocytes (arrow)at the posterior pole. (H) Whole-mount of a second adult retinasubjected to triple-label immunohistochemistry as in (G). GFAP⁺astrocytes did not express Pax2. Color versions of this photograph areavailable from the patentee.

ABBREVIATIONS

ABBREVIATION DESCRIPTION APCs astrocyte precursor cells IPAs immatureperinatal astrocytes MPAs mature perinatal astrocytes GFAP glialfibrillary acid protein; expressed in all astrocyte cells except APCsCNS central nervous system FACS fluorescence activated cell sorter Pax2marker expressed in APCs, IPAs and MPAs and a proportion of adultastrocytes vimentin marker expressed in APCs and IPAs DTPAdiethylenetriaminepentacetic acid EDTA ethylenediaminetetracetic acid

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the use of markers toselectively enrich lineage-specific cells from mammalian brains. Themammalian brains may be from a prenatal stage (e.g. an embryo) or from apostnatal animal including an adult. It is particularly significant thatAPCs have been identified, in accordance with the present invention,from adult brains. Reference herein to a brain includes parts thereofsuch as the retina or parts thereof. Even more particularly, the markersdistinguish the four developmental stages of astrocyte maturation whichare astrocyte precursor cells (APCs), immature perinatal astrocytes(IPAs), mature perinatal astrocytes (MPAs) and adult amd agedastrocytes.

Although in many circumstances, it is desirable to use immature cells,i.e. APCs and IPAs, the present invention extends to the use of allastrocyte types including MPAs and adult astrocytes.

In accordance with the present invention, the four stages of astrocytedevelopment may be characterized by differential marker expression asdescribed below in Table 1:— TABLE 1 Astrocyte cell types Marker APCsIPAs MPAs Adult astroycte Aged astrocyte Pax2 + + + −/+ −/+ GFAP− + + + + vimentin + + − − − S-11 − + + + +

Accordingly, one aspect of the present invention contemplates a methodfor generating a substantially homogeneous population oflineage-specific cells from tissue of the central nervous system (CNS)of mammalian animals, said method comprising subjecting said CNS tissueto tissue disruptive means to provide a mixed population of cellscomprising the lineage-specific cells to be isolated, subjecting themixed population to cell separation discrimination means to generate asubstantially homogeneous population of lineage-specific cells.

A number of cell isolation, cell separation, and cell purging strategiesare known for purifying or removing cells from a suspension comprising adiverse population of cells. Cell separation methods that are used toisolate cells or purge cell suspensions or cell populations typicallyfall into one of three broad categories. Physical separation methodstypically exploit differences in a physical property between cell types,such as cell size or density (e.g. centrifugation); chemical-basedmethods typically employ an agent that selectively kills or purges oneor more undesirable cell types; and affinity-based methods typicallyexploit antibodies or molecules with a selective binding capacity thatbind selectively to marker molecules on or in a cell membrane surface oron or in a cell of desired or undesired cell types, which antibodies maysubsequently enable the cells to be isolated or removed from thesuspension. It is not intended that the method of purification of cellsof astrocyte precursor cells be limited to any one method. However, cellmarker separation means is the most convenient to date and, inparticular, sorting of cells by immunological recognition of cellmarkers.

Once the starting source of CNS tissue is obtained, astrocyte cells canbe removed, and thus selectively separated and purified, by variousmethods which preferably utilize antibodies and cell markers. In thesemethods, antibodies or molecules that selectively bind to specificmarker molecules present on or in, for example, the astrocyte precursorcells of interest, but do not bind to other cells within the sourcematerial. The bound molecule then acts as a flag to signal theidentification of the appropriate cell type.

Cell types of non-astrocytic lineage can be removed using negativeselection (e.g. by immunopaning) as follows: N-CAM (PSA-N-CAM), 01, 04,GlC, Gal-C, NG2 and anti-mog for removal of oligodendrocytes and theirprecursors; CD31 and CD34 for removal of vascular endothelial cells andother markers for removal of fibroblasts.

These techniques can include, for example, flow cytometry using afluorescence activated cell sorter (FACS) and specific fluorochromes,biotin-avidin or biotin-streptavidin separations using biotin conjugatedto cell marker-specific antibodies and avidin or streptavidin bound to asolid support such as affinity column matrix or plastic surfaces ormagnetic separations using antibody-coated magnetic beads. Reference to“astrocyte cells” includes reference to all forms of cells includingAPCs, IPAs, MPAs and adult astrocytes and aged astrocytes. These are allencompassed by reference to astrocytic lineage. Depending on thecondition to be treated, the selection of which type of astrocyte cellcan be made. Astrocyte cell hybrid may also be employed using differentastrocyte cells or astrocyte cells with different neural cells.

In an alternative method, which is particularly efficient for thepractice of the present invention, a negative selection protocol isadopted. In a negative selection, the markers are on cell types ofinterest of astrocytic lineage. Consequently, non-astrocyte cells areremoved or astrocyte cells of not the desired level of maturity orimmaturity are removed.

Therefore, separation via cell marker discrimination utlilizesantibodies or other molecules that selectively bind specific markers andcan be achieved by negative or positive selection procedures. Innegative separation, antibodies are used which are specific for markerspresent on or in undesired cells, as for example, in the case of anastrocyte precursor population, where it would be desirable to depletethe number of non-precursor cells. In this case, antibodies could bedirected to the extracellular domain of proteins not present on or inthe precursor cells. Cell markers suitable for such a method of celldiscrimination include but are not limited to positive markers such asGD3 and A2B5 and negative markers such as GlC, 04, NG2. Pax2, GFAP andvimentin represent useful histochemical markers. Alternatively, it maybe desirable to directly select the desired cells from a population ofcells. In this case, antibodies or other molecules that selectively bindan extracellular domain of a cell protein can be used. Cells bound bysuch an antibody to a cell marker can then be sorted and the remainingcells removed and the desired mixture retained. Cell markers suitablefor such a method of cell discrimination include but are not limited toGD3 and A2B5.

The cell markers used for cell discrimination means may be labeled witha fluorescent compound. When the fluorescently labeled antibody ormolecule with selective binding capacity is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoeryirin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody or molecule with selective binding capacity can also bedetectably labeled using fluorescence emitting metals such as ¹⁵²Eu, orothers of the lanthanide series. These metals can be attached to theantibody or molecule with selective binding capacity using such metalchelating groups as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). The antibody also can bedetectably labeled by coupling it to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged antibody or molecule withselective binding capacity is then determined by detecting the presenceof luminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester. Likewise, a bioluminescent compound can be usedto label the antibody or molecule with selective binding capacity of thepresent invention. Bioluminescence is a type of chemiluminescence foundin biological systems, in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin. All such methods oflabeling an antibody or a molecule with selective binding capacity arecontemplated by the present invention.

The cell marker discrimination means includes contacting the cells to atleast one cell marker at least resident on the lineage-specific cells tobe isolated and using same to isolate such cell types and then usinganother molecule interactive with at least one other marker either onthe cell of interest or on cells to be discarded to enrich for thedesired lineage-specific cells.

The above method may be varied to use the first contact with aninteractive molecule to discard cells not intended to be isolated.

Tissue of the CNS includes the brain or parts thereof including theretina. The retina is considered part of the brain and is connected tothe brain via the optic nerve.

The lineage-specific cells particularly preferred in accordance with thepresent invention are astrocyte cells and, most preferably, are APCs orIPAs. However, any astrocyte cell may be isolated according to themethods of the present invention.

Accordingly, another aspect of the present invention provides a methodfor isolating APCs or IPAs from tissue of the CNS such as brainincluding retina tissue, said method comprising subjecting said CNStissue or part thereof to tissue disruptive means to provide a mixedpopulation of cells comprising the APCs or IPAs to be isolated,subjecting the cells to interactive molecules to a cell markerselectively present or absent on or in or in said APCs or IPAs togenerate a population comprising at least APCs and/or IPAs and thencontacting the isolated cells with an interactive molecule to at leastone other cell marker specific for either said APCs or IPAs or specificfor a cell marker absent from either APCs or IPAs to selectively removeor retain the desired cell type.

Where the desired cell types are APCs or IPAs, APCs are the mostpreferred. Other forms of astrocytes such as MPAs or adult astrocytesmay also be useful and are encompassed by the present invention. Fortissue replacement therapy, however, APCs or IPAs may be used but APCsare most preferred.

The selection of particular cell types is based on the differentialexpression of cell markers and, in particular, those selected from Pax2,GFAP, S-100 and vimentin in or on APCs, IPAs, MPAs and adult astrocytesand aged astrocytes as described in Table 1.

The preferred interactive molecules are immunoglobulins and are referredto herein as immunointeractive molecules. The term “immunoglobulins”encompasses antibodies or antigen-specific binding portions thereof suchas Fab fragments. The term “immunoglobulins” or “antibodies” furtherencompasses synthetic or recombinant or hybrid forms of these molecules.

Accordingly, in a preferred embodiment, the present inventioncontemplates a method of generating a substantially homogeneouspopulation of APCs from tissue of the CNS such as from brain or partsthereof including the retina or parts thereof, said method comprisingsubjecting said CNS tissue to cell disruptive means to produce apopulation comprising APCs amongst other cells and subjecting saidpopulation of cells to specific immunological separation using aGD3⁺-positive and/or A2B5⁺-positive selection technique and then anegative selection comprising GlC, 01, 04, Gal-C, NG2 and/or anti-Mogcells to generate a population of APCs. In a preferred embodiment,following the positive selection, the cells are cultured in a suitableculture medium such as serum free medium, for example, DMEM/F-12together with growth factors such as bFGF or chick embryo extracts. Theoligodendrocytes are then removed using the negative selection. Thepurity of these cells is determined and/or confirmed using a combinationof a Pax2, vimentin, GFAP and S-100 immunohistochemically. APCs arePax2⁺, GFAP⁻, S-100⁻ and vimentin⁺. One skilled in the art willimmediately recognize that other markers may be employed and all suchdifferentiating markers are encompassed by the present invention.

In another preferred embodiment, the present invention is directed to amethod of generating a substantially homogeneous population of IPAs fromthe APCs described above by exposing said APCs to CNTF, LIF, BMPincluding BMP4, TGFβ, cAMP and/or EGF to induce GFAP expression anddifferentiation along the maturation pathway.

Given the range of differential marker expression on astrocyte cells,one skilled in the art will readily recognize the ability to selectspecifically any cell type such as APCs, IPAs, MPAs or adult astrocytes.Furthermore, there may be a number of alternative combinations of cellmarkers which would be equally efficacious in isolating the one desiredcell type.

Reference herein to a “population” of cells means two or more cells. A“homogeneous population” means a population comprising substantiallyonly one cell type. A “cell type” may be cells of the same lineage orsub-type having substantially the same physiological status. Preferredhomogeneous populations comprise substantially only APCs or IPAs or MPAsor adult astrocytes or aged astrocytes. In terms of tissue replacementor augmentation therapy, the cells may be derived from the subject to betreated (autologous therapy) or from a suitably histocompatibilitymatched undivided (heterologous or non-autologous therapy).

Reference herein to a “substantially homogeneous population” refers to acell population in which a substantial number of the total population ofthe cells are of the same type and/or are in the same state ofdifferentiation. Preferably, a “substantially homogeneous population” ofastrocyte cells comprises a population of cells of which at least about50% are of the same cell type (e.g. APCs, IPAs, MPAs or adultastrocytes), more preferably that at least about 75% are of the samecell type, even more preferably at least about 85% are of the same celltype, still even more preferably at least about 95% of the cells are thesame type, and even more preferably at least about 97% (e.g. 98%, 99% or100%) are of the same cell type.

The term “tissue-disruption means” includes dissociation of individualcells from the connecting extracellular matrix (ECM) of the CNS tissue.Preferably, a single cell suspension is produced.

The preferred cells are generally not fully differentiated and, hence,may be regarded as committed (i.e. single lineage) but neverthelesspartially undifferentiated.

“Undifferentiated” means a primordial state of a cell or cells capableof differentiation and proliferation to produce progeny cells that canbe physiologically, biochemically, morphologically, anatomically,immunologically, physiologically, or genetically distinct from theprimordial state. The preferred undifferentiated cells are APCs or IPAs.These cells are capable of differentiation or maturation into MPAs andthen adult astrocytes.

The present invention is directed to CNS from mammalian subjects. Suchsubjects include primates, humans, livestock animals (e.g. sheep, cows,horses, donkeys, goats, pigs), laboratory test animals (e.g. mice, rats,rabbits, guinea pigs, hamsters) and companion animals (e.g. dogs, cats).Preferred animals are humans and laboratory test animals such as ratsand mice.

The CNS may be disrupted in vitro and then subjected to immunologicalseparation of particular cells and/or may be immobilized in a solidphase such as frozen sections and/or a gelatin matrix.

In one particular embodiment, CNS tissue is subjected to cryostattreatment and sections cut and mounted onto gelatin coated shades. Thesections may then be subjected to immunological testing with individualor combinations of antibodies.

The antibodies contemplated for use in accordance with the presentinvention may be prepared in any animal such as rabbit, mouse, rat,guinea pig, horse, sheep, pig, amongst a range of other animals orbirds, such as chickens or other poultry birds. The antibodies areconveniently directed to synthetically prepared or recombinantlyproduced or naturally occurring, purified forms of Pax2, GFAP, S-100 orvimentin. Furthermore, structurally or antigenically related moleculesmay also be employed which elicit antibodies which cross-react with oneof Pax2, GFAP, S-100 and vimentin.

The present invention is predicated in part on the use of positive andnegative cell selection of cell surface markers such as subjecting cellsto positive selection using surface markers GD3, A2B5, C3B2, FGFR3and/or PDGFRA or a combination thereof, then subjecting the positivelyselective cells to negative selection using GlC, O4 and/or NG2 or acombination thereof. The identity of the purified population of cells isconfirmed using Pax2, vimentin, GFAP and S-100 immunohistochemically.

The present invention provides, therefore, a substantially homogeneouspopulation of mammalian lineage-specific cells from the CNS, said cellsmade by the method comprising subjecting said CNS tissue to tissuedisruptive means to provide a mixed population of cells comprising thelineage-specific cells to be isolated, subjecting the mixed populationto cell separation discrimination means to generate a substantiallyhomogeneous population of lineage-specific cells.

More particularly, the present invention is directed to a substantiallyhomogeneous population of APCs or IPAs from the CNS such as brainincluding retina tissue prepared by the method comprising subjectingsaid CNS tissue or part thereof to tissue disruptive means andcontacting said immobilized tissue with interactive molecules to a cellmarker selectively present or absent on or in said APCs or IPAs togenerate a population comprising at least APCs and/or IPAs and thencontacting the isolated cells with an interactive molecule to at leastone other cell marker specific for either said APCs or IPAs or specificfor a cell marker absent from either APCs or IPAs to selectively removeor retain the desired cell type.

Preferably, the homogeneous population of cells comprises substantiallyonly APCs or IPAs, prepared as described above.

The ability to generate a substantially homogeneous population ofparticular astrocyte cells such as APCs or IPAs permits the developmentof a range of therapeutic applications such as tissue replacementtherapy, augmentation therapy (for example, co-transplantation of humanstem cells with human APCs and/or IPAs) and therapy to repair, replicateor delay senescence of astrocytes.

Accordingly, another aspect of the present invention contemplates amethod of cell replacement therapy in a mammalian animal, said methodcomprising generating a substantially homogeneous population oflineage-specific cells and introducing same into an organ or tissuerequiring cells to be replaced or to another location from where thecells can migrate to an organ or tissue requiring cells wherein theintroduced cells are subject to expansion or proliferation in vitroand/or in vivo by one or more growth factors.

Examples of suitable growth factors include inter alia CNTF, LIF, BMPsuch as BMP4, TGFβ, cAMP and EGF.

More particularly, the present invention contemplates a method of cellreplacement or augmentation therapy in a mammalian animal, said methodcomprising generating a substantially homogeneous population ofastrocyte precursor lineage-specific cells and introducing same into anorgan or tissue requiring cells to be replaced or to another locationfrom where the cells can migrate to an organ or tissue requiring cellswherein the introduced cells are subject to expansion or proliferationin vitro and/or in vivo by one or more growth factors.

Preferably, the astrocyte precursor is APCs or IPAs.

This method of the present invention is useful for treating a range ofdegenerative disorders including Alzheimer's disease, HIV-associateddementia (HIVD), Huntington's disease, chronic neurological disorders,Parkinson's disease, epilepsy, stroke or alcoholism. Furthermore, thepresent method may be used to treat hypoxia or the effects thereof aswell as spinal cord injuries. Other conditions contemplated fortreatment by the present invention include acute brain injury (e.g. headinjury or cerebral palsy) and a large number of CNS dysfunctions (e.g.depression and schizophrenia). In recent years, neurodegenerativedisease has become an important concern due to the expanding elderlypopulation which is at greatest risk for these disorders. Thesediseases, which include Alzheimer's disease, multiple sclerosis (MS),Huntington's disease, amyotrophic lateral sclerosis and Parkinson'sdisease, have been linked to the degeneration of cells in particularlocations of the CNS, leading to the inability of these cells or thebrain region to carry out their intended function. The treatment of allsuch conditions is encompassed by the present invention. Otherconditions contemplated herein include Angleman sydrome,Charcot-Marie-Tooth disease, epilepsy, essential tremor, fragile Xsyndrome, Friedreich's ataxia, Niemann-Pick disease, Prader-Willisyndrome, Rett syndrome, spinocerebella atrophy and William's syndromeas well as other conditions affecting the brain or CNS such as a stroke,alcoholism or drug or other substance abuse. Furthermore, visual and/orcognitive impairment due to, for example, aging dementia may also betreated. Still further, the method of the present invention is usefulfor augmentation therapy to regenerate aging tissue includingco-transplantation of human APCs and/or IPAs with human stem cells (e.g.neural stem cells). The treatment of these conditions such as HIVD inaccordance with the present invention is particularly relevant due tothe demonstrated effect of astrocytes on these conditions and inparticular HIVD.

In accordance with the present invention, astroycte precursors such asAPCs or IPAs are collected from a suitable source and homogeneouspopulations prepared using marker discrimination means as describedabove. At this point, the cells may be optionally frozen and stored forsubsequent use. Alternatively, or after storage, the cells are expandedin vitro by the use of one or more growth factors and from about 10⁵ toabout 10¹⁰ cells administered directly to the site affected on the brainor part thereof (e.g. retina) or other part of the CNS. Alternatively,or in addition, the cells may be administered to another part of thebrain or CNS where they migrate to the site required since APCs and IPAsretain their migratory potential.

In vitro expansion is a particularly convenient form of expansion but asan alternative or in addition to in vitro expansion, the one or moregrowth factors may be administered to the brain or other part of the CNSto facilitate APCs or IPAs expanding in vivo.

Reference herein to “cell replacement therapy” includes, in one form, aprocess in which undifferentiated APCs and/or IPAs are strategicallyplaced in vivo or in vitro such as to differentiate and proliferate intoa mature form of astrocyte. Thus, cell replacement therapy requires thatan undifferentiated astrocyte precursor cell appropriately differentiatefor the purposes of providing repair, regeneration or replacement of acell function. “Cell replacement therapy” also includes augmentationtherapy. The latter includes the removal of existing cells or tissue,expanding in culture and then replacing. This is a particular advantageof the present invention, where a single or a few astrocyte precursorcells are capable of expansion in culture to give rise to a large numberof astrocytes. The subject into which the purified astrocyte precursorcells are implanted for the purpose of “cell replacement therapy” orrepair of tissue, or from which stem cells can be derived, is preferablyan animal including but not limited to animals such as cows, pigs,horses, chickens, cats, dogs and is preferably a mammal such as aprimate and most preferably a human.

Furthermore, the essence of this aspect of the present invention is theco-transplantation of APCs and/or IPAs with neural stem cells to enhancethe proliferative, differentiative and/or maturation of both lineages.The latter includes neuronal stem cells. The former includes variousgrowth factors. The term “co-introduction” or “co-introduced” includesthe simultaneous or sequential administration of both the astrocytes andother cell or factor.

The present invention further contemplates using the astrocyte cellmarkers in a range of diagnostic applications in addition to using themarkers to selectively isolate astrocytes at a particular level ofmaturity. For example, in accordance with the present invention it hasbeen elucidated that Pax2 is no longer expressed or is only poorlyexpressed in a proportion of adult astrocytes and in a larger proportionof aged astrocytes. However, certain disease conditions or ageing mayresult in adult astrocytes beginning to express Pax2. The identificationof Pax2 expression in adult astrocytes and aged astrocytes may beindicative of particular disease condition, neurological dysfunction,level of ageing or a propensity to develop any of the latter conditions.

Accordingly, the present invention contemplates a method for assessingthe level of healthy tissue in a CNS biopsy such as a brain biopsy in anadult subject said method comprising determining in said biopsy presenceof Pax2⁺ astrocyte cells wherein the presence of said Pax2⁺ cells isindicative of a reversion in the maturation of said astrocytes.

As indicated above, however, there are a range of markers which can beused to characterize the astrocyte cell populations.

The identification of new growth factors for APCs and IPAs is furthercontemplated by the present invention. In one embodiment, cells arecultured in vitro and the culture supernatant tested using, for example,microarray technology or the cells themselves tested for differentialgene expression between different stages of maturation.

Suitable sources of astrocyte precursors include embryo or fetal brainincluding retinal tissue or other CNS tissue. As stated above, theisolated cells may be used immediately, subject to expansion in vitroand/or stored for subsequent use. The cells may be from the same subjectbeing treated (autologous therapy) or a different subject(non-autologous therapy). Autologous therapy is preferred.

In one embodiment, therefore, there is provided a composition ofastrocyte precursor cells such as APCs or IPAs in substantiallyhomogeneous form, said composition optionally further comprising one ormore pharmaceutically-acceptable carriers and/or diluents.

Methods for preparing cell compositions are well known in the art. Thecomposition of cells may also be stored in vials or other convenientcontainer means. The containers may also be in package form withinstructions for use.

As stated above, the present invention extends to growth factors fromastrocyte precursors.

These may be identified by any number of techniques including microarraytechnology. The latter is useful for identifying growth factor orautocrine factor receptors on the surface of particular astrocytes. Thiswill then enable selection of particular growth or autocrine factorswhich will facilitate differential and/or proliferation of particularastrocyte cells.

Furthermore, differential hybridization is another useful technique foridentifying other markers including growth or autocrine factor receptormarkers or particular astrocyte cells.

Another aspect of the present invention is directed to conditionedmedium from the in vitro culture of astrocyte precursor cells such asAPCs or IPAs wherein said conditioned medium comprises one or moregrowth factors or autocrine factors.

More particularly, the present invention contemplates a growth orautocrine factor obtainable from conditioned medium of an in vitro cellculture of astrocyte precursors such as APCs or IPAs. Examples of growthand autocrine factors include CNTF, LIF, BMP, TGF-β and EGF.

In an alternative embodiment, the present invention provides a growthfactor or autocrine factor identified by microarray technology ofastrocyte cells.

The growth or autocrine factor may be in isolated form or may be incomposition form such as comprising one or morepharmaceutically-acceptable carriers and/or diluents. This factor may beadministered directly to the brain or retina or other suitable locationto facilitate growth development of replacement tissue. This isparticularly possible since, in accordance with the present invention,Pax2⁺ GFAP⁻ APCs and Pax2⁺ GFAP⁺ astroctyes have been identified in thesub-ventricular zone of the lateral ventricle of the adult brain. Incontrast, no Pax2⁺ GFAP⁻ APCs have been identified in thesub-ventricular zone of the third ventricle where GFAP⁺ Pax2⁻ adult/agedastrocytes have been identified.

The present invention further contemplates the use of the purifiedastrocytes and in particular astrocyte precursor cells as gene therapycarriers. According to this embodiment, genetic material, encodingneurobiologically-useful factors, is introduced into the astrocytesprior to administration to a subject. Neurobiologically-useful factorscontemplated by the present invention include growth factors, cytokines,proliferation and/or differentiation promoting agents and anti-viral andother anti-pathogenic agents. In accordance with this aspect of theinvention, genetic material encoding these factors is cloned into avariety of vectors including viral vectors and introduced into thecultured astrocytes. After appropriate selection, including optionallystable integration of the genetic material into the chromosome of theastrocytes, these can then be used in tissue repair or augmentationtherapy.

The present invention further provides a system of lineage-specific cellisolation from tissue of the CNS by the method of:—

-   (1) obtaining a sample from the CNS;-   (2) separating the cells from the CNS on the basis of cell surface    markers;-   (3) selecting cells having desired cell surface markers and making    these available for autologous or non-autologous transplantation;    and-   (4) optionally co-administering the isolated cells with neural    cells, neural stem cells or mature astrocytes or autocrine factors.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Collection, Age Determination and Preparation of Human CNSTissue

Human fetal eyes, ranging in age from 8 to 32 weeks gestation were usedin accordance with the guidelines set forth in the Declaration ofHelsinki. Fetuses older than 20 weeks gestation had died of naturalcauses after premature or difficult deliveries. Younger fetuses wereobtained after water bag- or prostaglandin-induced abortions, which arepermitted up to 20 weeks gestation. Embryonic or fetal brain tissue alsoprovided a useful source of cells. The age of each fetus was determinedfrom charts of crown-rump length and crown-heel length (Potter, E. L.and Graig, J. M., Pathology of the Fetus and the Infant, pp. 29-37,Yearbook Medical Publishers, Chicago, 1975). Three adult human retinaswere obtained from an eye bank and originated from individuals aged 69,69, and 79 years; the latter individual had a history of lung carcinomawhereas the other two had no significant medical history.

For preparation of retinal whole-mounts, the anterior segment andvitreous of each eye were removed and the eyecup was fixed with 4% w/vparaformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for a minimumof 2 days at 4° C. The retina was dissected as previously described (27)and permeabilized by washing for 30 min in phosphate-buffered saline(PBS) containing 1% v/v Triton X-100. After blocking of nonspecificbinding sites by incubation for 1 h with PBS containing 1% v/v bovineserum albumin, whole-mounts were exposed to primary antibodies.

For cryostat sections, the eyeball was washed several times in PBS andthen incubated at 4° C. in 25% w/v sucrose overnight, embedded in OCTcompound (Miles, Elkhart, Ind.), and rapidly frozen in isopentane-cooledliquid nitrogen. Cryostat sections (20 μm) were cut at −20° C. andmounted on gelatin-coated slides.

Whole-mounts of retinas from fetuses at various ages and from adults, aswell as cryostat sections of fetal and adult eyes, were examined withvarious combinations of antibodies to identify APCs and perinatalastrocytes. The antibodies were as follows: (i) rabbit polyclonalantibodies to Pax2 (1:100 dilution) (Babco), which were generated inresponse to a recombinant protein containing amino acids 188 to 385 ofPax2 and which react with the Pax2 proteins of a variety of species,including mouse, rat, human, chicken, Xenopus, and zebrafish; (ii) mousemonoclonal antibody (mAb) GA5 to GFAP (IgG1; 1:100 dilution) (Sigma);and (iii) mouse mAbs 3B4 (IgG2A; 1:4 dilution) (Boehringer Mannheim) andLN-6 (IgM; 1:200 dilution) (Sigma) to vimentin. To determine therelation between Pax2 immunoreactivity and the developing vasculature,the inventor labeled some retinas with both anti-Pax2 and the mouse mAbQBEND/10 to CD34 (IgG1; 1:50 dilution) (Serotec). Retinas were alsolabeled with both anti-CD34 and rabbit polyclonal antibodies to GFAP(1:2 dilution) (Biogenex) to determine the relation between astrocytedifferentiation and blood vessel formation, as previously described(Hughes et al., 2000, supra).

The glial population obtained following positive selection with markerssuch as A2B5, GD3, 3CB2, FGFR3 and PDGFRA are cultured in a serum freemedium such as DMEM/F-12 together with growth factors. These growthfactors include bFGF and chick embryo extract.

EXAMPLE 2 Pax2-GFAP, Pax2-vimentin, Pax2-CD34, and CD34-GFAPDouble-Label Immunohistochemistry

Retinal whole-mount preparations were incubated for 2 to 3 days at 4° C.with a mixture of anti-Pax2 and either mouse anti-GFAP, anti-vimentin,or anti-CD34. They were then washed three times with PBS containing 0.1%v/v Triton X-100, incubated for 4 h with a mixture of Cy3-conjugatedgoat antibodies to rabbit IgG (1:200 dilution) (Jackson ImmunoResearch)and fluorescein isothiocyanate (FITC)-conjugated sheep antibodies tomouse Ig (1:50 dilution) (Amersham), and washed three times with PBScontaining 0.1% v/v Triton X-100. For confocal microscopic analysis ofdouble labeling with anti-CD34 and rabbit anti-GFAP, retinas wereincubated overnight at 4° C. with the primary antibodies, washed andthen incubated with appropriate secondary antibodies as described above.For light microscopy, retinas were labeled with polyclonal anti-GFAP asdescribed previously (Hughes et al., 2000, supra) and then withanti-CD34. Labeling with anti-CD34 was detected by incubation at roomtemperature first for 4 h with biotinylated secondary antibodies (1:50dilution) (Amersham) and then for 2 h with alkalinephosphatase-conjugated Extravidin (1:100 dilution) (Sigma); the alkalinephosphatase reaction was performed with Fast Red tablets (Sigma).Whole-mounts were mounted in glycerol-PBS (1:2) with the nerve fiberlayer (NFL) uppermost, with the exception that, for examination of theventricular zone, they were mounted with the retinal pigment epithelium(RPE) uppermost.

Cryostat sections were allowed to air-dry for 15 min, after whichnon-specific binding sites were blocked by incubation for 30 min withPBS containing 1% v/v bovine serum albumin. Sections were then incubatedovernight at 4° C. in a humidified atmosphere with various combinationsof primary antibodies, washed three times with PBS, and incubated for 2h with appropriate secondary antibodies as described above. The sectionswere finally washed several times with PBS and mounted in glycerol-PBS(1:2).

EXAMPLE 3 Triple-Label Immunohistochemistry for Pax2, Vimentin and GFAP

For triple labeling, retinal whole-mounts and sections were incubatedovernight at 4° C. in a humidified atmosphere with a mixture ofanti-Pax2 and anti-vimentin. After washing three times with PBS, theywere incubated for 2 h with Cy3-conjugated anti-rabbit IgG and eitherFITC-conjugated goat anti-mouse IgG2a (1:50 dilution) (SouthernBiotechnology Associates) or Texas red-conjugated goat anti-mouse IgM(1:50 dilution) (Vector). The tissue was washed three times with PBS,incubated overnight at 4° C. with mouse anti-GFAP, washed three timeswith PBS, and incubated for 4 h with biotinylated goat anti-mouse IgG1(1:50 dilution) (Southern Biotechnology Associates). After washing threetimes with PBS, the tissue was incubated overnight at 4° C. withAMCA-conjugated streptavidin (1:100 dilution) (MDA Pharma) orCy5-conjugated streptavidin (1:100 dilution) (Jackson ImmunoResearch),washed with PBS, and mounted in glycerol-PBS (1:2).

EXAMPLE 4 Confocal Microscopy

Confocal microscopy was performed with a Leica argon-krypton lasermounted on a Leica Axiophot epifluorescence photomicroscope. FITC, Cy3,and Cy5 fluorescence was excited at 488, 588, and 665 nm, respectively.Images were collected at a resolution of 300 pixels per inch andprocessed with Adobe Photoshop V5.0 and Adobe PageMaker V6.5 software.

EXAMPLE 5 Mapping and Determination of Retinal Area and Cell Density

The outer limits of immunoreactivity and retinal outlines weredetermined with a Leica fluorescence microscope (model AH BT, attachmentmodel AH2-RFL) with a 10× eyepiece containing a 1-mm grid (Chan-Ling etal., Current Eye Res. 9: 459-478, 1990; Chang Ling, T., Microsc. Res.Techniq. 36: 1-16, 1997). The maps were scanned with an XRS-OmniMedia-3cx flatbed scanner and processed with the use of Adobe Photoshop V5.0software.

EXAMPLE 6 Specificity of Pax2 Expression to Astrocytic Lineage Cells inthe Developing Human Retina

With the exception of a small region at the ventricular zone surroundingthe ONH (see below), Pax2 expression was restricted to somas in the GCLand NFL of the developing human retina, as revealed by the retina at 24to 26 weeks gestation shown in FIG. 1A. Triple-labelimmunohistochemistry with retinal whole-mounts showed that anti-Pax2labeled only cells that were positive for the astrocytic lineage markersvimentin or GFAP (FIGS. 1, B and C). Double labeling with anti-CD34 andanti-Pax2 revealed that Pax2 is not expressed by endothelial cells.Three populations of Pax2⁺ cells were identified in the developingretina: (i) cells that were Pax2⁺, GFAP⁻, and vimentin⁺ (FIGS. 1, B andC) were designated APCs; (ii) cells that were Pax2⁺, GFAP⁺, andvimentin⁺ (FIG. 1B) were designated immature perinatal astrocytes; at 12weeks gestation, most GFAP⁺ cells were vimentin⁺; and (iii) cells thatwere Pax2⁺, GFAP⁺, and vimentin⁻ (FIG. 1, D through F) were designatedmature perinatal astrocytes; at 32 weeks gestation, most GFAP⁺astrocytes were vimentin⁻. Thus, the transition from an APC to animmature perinatal astrocyte in vivo is characterized by the onset ofexpression of GFAP, and the transition from immature to mature perinatalastrocytes is characterized by the loss of expression of vimentin.

To distinguish astrocytes of the fetal and neonatal human retina fromthose present in the adult CNS, the inventor has adopted the termperinatal astrocytes, as used by Mi and Barres (1999, supra), throughoutthis study, even though these cells are already present in the embryonicretina. A similar terminology has been applied to cells of theoligodendrocyte lineage (Wolswijk, G. and Nobel, M., Development 105:387-400, 1989), in which perinatal and adult oligodendrocyte precursorcells exhibit differences in such characteristics as cell cycle time,proliferative capacity and rate of migration.

A summary of markers expressed during the different developmental stagesof the astrocytes is shown in Table 1.

EXAMPLE 7 Morphology and Location of APCs and Perinatal Astrocytes:Relations with Ganglion Cell Axons and Forming Blood Vessels

Pax2⁺, GFAP⁻ APCs were located superficially, adjacent to the innerlimiting membrane, and were characterized by a predominantly sphericalor ovoid morphology with a soma diameter of ˜12 to 25 μm (arrows in FIG.1, A through C). APCs migrated superficially over regions of the retinacontaining immature perinatal astrocytes (arrows in FIG. 2, A, B, andD). At 24 to 26 weeks gestation, perinatal astrocytes were abundant inthe central region of the retina (FIG. 2, A through C), whereas onlyPax2⁺, GFAP⁻ APCs were evident more peripherally (FIG. 2, D and E). Atthe edge of the retina, no Pax2⁺ cells were evident at this time (FIG.2F). Most immature perinatal astrocytes at the leading edge of GFAPimmunoreactivity exhibited an approximately spherical soma, with adiameter of 6 to 10 μm, and possessed bipolar GFAP⁺ processes locatedsuperficially in the nerve fiber layer (NFL) (FIG. 1D). Mature perinatalastrocytes located more centrally in the NFL adopted a morphologycharacterized by multiple parallel processes and were closely alignedalong nerve fiber bundles (NFBs) (FIG. 1G), whereas those located in thedeeper ganglion cell layer (GCL) adopted a predominantly stellatemorphology with numerous processes ensheathing blood vessels (FIG. 1E).Mature perinatal astrocytes exhibited an ovoid soma with a diameter of 6to 10 μm. Consistent with previous observations (Provis, 1997, supra;Hughes et al., 2000, supra), the outer limit of astrocyte migration intothe human retina preceded the outer limit of patent vessels visualizedby anti-CD34 immunohistochemistry (FIG. 1H).

EXAMPLE 8 APCs and Perinatal Astrocyte Differentiation at the ONH

Retinal astrocytes immigrate from the optic nerve (Ling and Stone, 1988,supra; Watanabe, T. and Raff, M. C., Nature 332: 834-837, 1988; Ling etal., 1989, supra; Huxlin et al., J Neurocytol. 21: 530-544, 1992). Thepathway of invasion of cells of the astrocytic lineage is, therefore,thought to lead from the ONH into the peripheral retina. The inventorapplied double-label immunohistochemistry for Pax2 and GFAP to cryostatsections of eyes at 8, 12, 16, and 24 to 26 weeks gestation in order toexamine the distribution of APCs and perinatal astrocytes at the ONHduring development. Consistent with the previous demonstration of Pax2gene expression in the region of the human optic disk and optic nerve(Terzic et al., 1998, supra), the inventor detected Pax2immunoreactivity in the optic nerve and at the ONH at 8 weeks gestation(FIG. 2, G and H). Quantitative analysis of such a double-labeledsection of the human ONH at 8 weeks gestation revealed that 34% of Pax2⁺cells were Pax2⁺, GFAP⁻ APCs, with the remainder being Pax2⁺, GFAP⁺perinatal astrocytes.

FIG. 2G is a photographic montage of the ONH region at 8 weeks gestationand FIG. 2H is a schematic representation of this region showing theprecise locations of individual APCs and perinatal astrocytes. GFAP⁺perinatal astrocytes had reached the distal limit of the human opticnerve, and many of these cells had extended into the ONH. APCs were nolonger evident at the retinal end of the optic nerve, but were dispersedthroughout the ONH.

EXAMPLE 9 APCs and Perinatal Astrocytes at the Ventricular Zone of theFetal Retina

Transverse sections revealed a cluster of Pax2⁺ somas present in a smallregion surrounding the ONH at the ventricular surface of the humanretina (FIG. 2, G through J). This cluster of Pax2⁺ somas was located atthe innermost margin of the ventricular zone of the retina, at itsjunction with the numerous optic nerve axons that exit the retina toform the optic nerve. The presence of these cells and the extent oftheir distribution were conformed by examining the ventricular surfaceof retinal whole-mounts at various ages.

The morphology of these cells in the peripapillary region of the retinaat 14 to 16 weeks gestation and 32 weeks gestation is shown in FIG. 3 (Athrough D). Two populations of Pax2⁺ cells were discerned at theventricular zone: Pax2⁺, vimentin⁺, GFAP⁺ immature perinatal astrocyteslocated in the region surrounding the ONH, and Pax2⁺, vimentin⁺, GFAPAPCs located predominantly at the periphery of the Pax2⁺ region. Theextent of the distribution of perinatal astrocytes as well as theprecise locations of individual Pax2⁺ APCs at the ventricular surface ofthe retina at various fetal ages are shown in FIG. 3 (E through G).

EXAMPLE 10 Timing and Topography of APC and Perinatal Astrocyte Invasionof the Retina

Pax2⁺, GFAP⁻ APCs were consistently detected in a small region ahead ofthe Pax2⁺, GFAP⁺ perinatal astrocytes during development of the humanretina (FIG. 4). Triple labeling confirmed that all cells at the leadingedge were Pax2⁺, vimentin⁺, GFAP⁻ APCs (FIG. 5, A and B). Even at theyoungest age examined with transverse sections (8 weeks gestation), APCshad extended ˜560 μm from the ONH, and, with increasing maturation, theouter limit of their distribution expanded (FIG. 4). At each stage ofmaturation, the outer limit of perinatal astrocytes lagged behind thatof APCs. The spread of both APCs and perinatal astrocytes was centeredon the ONH, and it showed an approximately four-lobed topography earlyin embryonic development (FIG. 4). APCs and differentiated astrocytesreached the edge of the retina by 28 weeks gestation and between 28 and32 weeks gestation, respectively. APCs persisted at reduced densitiesthroughout the retina at 32 weeks gestation, the oldest fetal ageexamined.

APCs and perinatal astrocytes followed a curved pattern of migration inthe temporal retina, mimicking the pattern of NFBs and blood vessels inthe human retina. The density of APCs and perinatal astrocytes wasmarkedly reduced in the raphe region. Throughout the observation period,neither APCs nor committed astrocytes were detected in the incipientfoveal zone (FIG. 4; FIG. 5, C through E), consistent with previousobservations (Ramirez et al., 1994, supra; Trivino et al., 1997, supra).However, individual isolated astrocytes were apparent in the perifovealregion at 18 weeks gestation (FIG. 5, C and D), and, at 18 weeksgestation, perinatal astrocytes were observed aligned along NFBs in theraphe region (FIG. 5, E and F).

EXAMPLE 11 Astrocytic Lineage in the Adult Human Retina

The inventor's observation that APCs are present in substantial numbersin the human retina at 32 weeks gestation prompted us to examine whethersuch cells also exist in the adult retina. The inventor appliedtriple-label (Pax2-GFAP-vimentin) immunohistochemistry to retinalwhole-mounts and transverse sections prepared from three adult humaneyes. Anti-Pax2 did not label neurons or endothelial cells in the adulthuman retina, and GFAP⁺ astrocytes showed a morphology and distributionconsistent with those previously described (Ramirez et al., 1994, supra;Trivino et al, 1997, supra). The inventor identified two distinctpopulations of astrocytic cells in the adult human retina: Pax2⁺, GFAP⁺,vimentin mature perinatal astrocytes (FIG. 5G) and Pax2⁻, GFAP⁺,vimentin⁻ adult astrocytes (FIG. 5H).

Topographical analysis of the distribution of these two types ofastrocytes revealed that Pax2⁺, GFAP⁺, vimentin⁻ mature perinatalastrocytes were restricted to the region surrounding the ONH, whereasPax2⁻, GFAP⁺, vimentin adult astrocytes were present throughout theretina with the exception of the foveal region (FIG. 4; FIG. 5, G andH). Thus, in the adult human retina, most cells of the astrocyticlineage no longer express Pax2. Because of the ability to detect smallnumbers of cells with the retinal whole-mount technique (Hu et al., Am.J. Pathol. 156: 1139-1149, 2000), the inventor mapped two whole-mountsto determine whether any APCs were present in the normal adult humanretina. Every Pax2⁺ soma was checked for surrounding GFAPimmunoreactivity, and all were found to be GFAP⁺. On the basis of thissystematic evaluation, it is concluded that no Pax 2⁺, GFAP⁻ APCs werepresent in the adult human retinas, which were derived from individualsover 65 years of age.

Although the inventor detected both APCs and committed astrocytes at theventricular zone in the region adjacent to the ONH during embryonicdevelopment, triple-label immunohistochemical analysis of transversesections failed to detect either of these cell types in this region ofthe adult human retina.

EXAMPLE 12 Implications for Optic Nerve Coloboma

The observation that Pax2 expression is specific to cells of theastrocytic lineage in the intact human fetal retina suggests thatcongenital optic nerve colobomas might be attributable to aberrantastrocytic differentiation in the ventricular zone during embryonicdevelopment. Colobomas result from imperfect formation or closure of thefetal cleft of the optic vesicle during embryogenesis.

Some human optic nerve colobomas are associated with abnormalities inPax2 gene expression (Sanyanusin et al., 1995, supra). In normal humanembryos, Pax2 mRNA is abundant in the optic nerve and ONH during theperiod of expected closure of the choroidal fissure (Terzic et al.,1998, supra). In mice with impaired expression of Pax2, the band ofPax2⁺ cells that surrounds the retinal ganglion cell axons as they exitthe retina becomes disorganized and the surrounding retinal tissue is nolonger clearly separated from the axons, resulting in dispersal of theaxons over a much wider region (Otteson et al., 1998, supra). Theinventor has now shown that the Pax2⁺ cells of this cuff in the humanretina comprise APCs and perinatal astrocytes, suggesting that Pax2⁺cells of the astrocytic lineage play a critical role both in delineatingthe axons of the ONH from the surrounding retinal tissue duringdevelopment and in funneling and restricting the pathway of axonal exitfrom the retina. Thus, optic nerve colobomas may be caused by aberrantdifferentiation of cells of the astrocytic lineage at the peripapillaryzone.

EXAMPLE 13 In Vivo Characterization of Type-1 APCs in Rat Retina

A developmental series of rat retinal whole-mount preparations from E15to adulthood is subjected to double-label IHC for Pax2 and GFAP. Type-1APCs is defined by the phenotype Pax2⁺ GFAP⁻, whereas committedastrocytes is defined by the phenotype Pax2⁺ GFAP⁺. The temporal andtopographical distributions of APCs and committed astrocytes aredetermined, as are the morphology of these cells and their associationswith neighbouring structures such as neurons and blood vessels. Animalsare anaesthetized with sodium pentobarbitone (60 mg/kg, i.p.) andperfused transcardially with phosphate-buffered saline containing 4% v/vparaformaldehyde. Retinal whole-mount preparations and transversesections are prepared as previously described.

Preliminary observations by the inventor have shown that APCs arepresent in the central rat retina for only a limited period (E16 toE20). Labeling with bromodeoxyuridine is also combined with Pax2 or GFAPIHC to determine the proliferative capacity of APCs and committedastrocytes in the retina at various stages of development. These studiesprovide important information on the morphology, antigenic phenotype,migration, and proliferation of astrocytes at various stages ofdifferentiation in vivo. Various astrocyte-specific markers, includingvimentin and S100, as well as vascular markers are also examined ascontrols.

EXAMPLE 14 Isolation, Purification, Expansion and ControlledDifferentiation of APCs from Embryonic Rat Retina

Regions of the central rat retina in which APCs are determined to beabundant are isolated from embryos on E17 to E18, the stage ofdevelopment at which the preliminary data indicate that the number ofAPCs is maximal. The tissue is dissociated according to standardprotocols and APCs are isolated by positive or negative immunopanning orfluorescence-activated cell sorting with the use of antibodies tospecific cell epitopes. Markers appropriate for negative selection ofvascular endothelial cells, microglia, oligodendrocytes and theirprecursors and neurons include the Griffonia simplicifolia (GS) lectin,C5, embryonic N-CAM, and the ED1 and 04 antibodies. Immunohistochemistrywill reveal a substantial population of cells which are Pax2⁺ and GD3⁺among a predominantly GFAP⁻ population, indicating that GD3 is anappropriate surface marker for APCs. Other positive selection markersinclude A2B5, 3CB2, PDGFRα and FGFR3. Purified populations of APCs arecultured in plates coated with either fibronectin alone or fibronectinplus laminin, and maintained in SATO defined medium supplemented withvarious growth factors both to facilitate their expansion and to inhibittheir differentiation into committed astrocytes. Growth factors testedalone and in combination include CEE, basic fibroblast growth factor,neurotrophin-3 and ciliary neurotrophic factor. Various conditions,including withdrawal of CEE and exposure to thyroid hormone, retinoicacid, ciliary neurotrophic factor, leukemia inhibitory factor, or bonemorphogenetic proteins, are also tested for their ability to inducedifferentiation of APCs.

EXAMPLE 15 Analysis of Gene Expression in Cells of the Astrocyte Lineage

The major obstacle of differential display is the issue ofdiscriminating between false positives and truly differentiallyexpressed mRNAs. This process is arduous and requires large amounts ofRNA. To overcome this problem, cDNA probes are generated from amplifiedRNA following standard protocols that allow differential screening ofmRNA species with a frequency as low as 1/40,000. RNA is extracted frompure, expanded populations of type-1 APCs and linearly amplified anestimated 10⁶-fold using T7 RNA polymerase amplification (Ampliscribe T7Transcription Kit, Epicentre Technologies, Madison, Wis.). Cy3-labeledcDNA synthesized from the amplified RNA is hybridized to rodentmicroarrays and analyzed using ScanArray 3000 reader (General Scanning,Watertown, Mass.). The pattern of gene expression is determined for theretinal APCs are compared with those of neuroepithelial stem cells, GRPcells, and neuron-restricted precursor cells of the spinal cord as wellas O-2A progenitor cells of rat optic nerve. The pattern of geneexpression is thus examined to identify Type I astrocyte-specific genesat various stages of differentiation.

EXAMPLE 16 Determination of the Developmental Potential of APCs In Vitroand In Vivo

APCs is subjected to clonal analysis by plating at a density of 10 to 50cells per dish. Single cells are identified after culture for 4 h,expanded in medium supplemented with appropriate growth factors for 4days, and induced to differentiate. Clonal plates are then subjected totriple-label cytochemical analysis of appropriate cell markers in orderto identify the progeny of the precursor cells. Cells at various stagesof differentiation are thus identified by their expression ofdevelopmental stage-specific markers.

In addition to these in vitro experiments, pure populations of APCslabeled with green fluorescent protein are injected into the vitreouschamber of adult rats or rat pups during the first week after birth withthe use of a 10-μl Hamilton-type microsyringe. Retroviral vectors andvirus preparation are then carried out. All studies are undertaken withinbred Fischer 344 rats to allow syngeneic transplantation. CulturedAPCs will be harvested with trypsin, washed and suspended at a densityof 100,000 cells with media containing 20 ng of bFGF. Initially, 10,000cells will be transplanted per eye in order to assess the survival andincorporation into the host tissue of the donor cells, which will beexamined after 21 days.

EXAMPLE 17 DNA Microarray

DNA microarray technology is used to identify markers and growth factorreceptors that are expressed at various stages of the astrocyte lineageduring development. Such information is fundamental to understanding thedevelopmental biology of these cells and facilitates isolation andexpansion of cell populations suitable for clinical application in therepair of degenerative neuropathies. In addition, characterization ofconditions that promote expansion of APCs renders feasible therecruitment of endogenous populations of these cells for tissue repair.

EXAMPLE 18 In Vivo Model

Populations of rat APCs labeled with GFP are implanted into the retinaof neonatal and adult rats and the fate of these cells examined in orderto assess their potential for integration into the normal developing andadult CNS. Studies designed to determine whether co-transplantation ofrat APCs with neural precursor cells facilitates neural repair by thelatter cells in various experimental models of CNS damage are conductedincluding the rat retina injured by transient ischaemia and the retinaof immature and mature dystrophic rats. Further, similar studies will beundertaken in the rat model of foetal alcohol syndrome. These variousstudies provide a source of human cells available to neurosurgeons fortransplantation in humans.

EXAMPLE 19 Characterization of Cells of the Astrocyte Lineage in HumanFetal, Adult and Aged Brains In Vivo

Sections of human brain derived from embryos and adults of various agesare subjected to triple-label immunohistochemical analysis withantibodies to Pax2, vimentin and GFAP, according to the protocolsdescribed above in order to characterize the antigenic phenotype, timingof differentiation, distribution and morphology of cells of theastrocytic lineage.

These studies complement the characterization of this cell lineage inthe human and rat retina (fetal, adult and aged) and provide importantinformation on the morphology, antigenic phenotype and distribution ofcells of the astrocytic lineage. Studies on human fetal brains definethe developmental period during which APCs can be isolated from thehuman fetal brain as well as the regions of the brain that provide thehighest yield of these cells. Furthermore, they will shed light on theregions of the normal adult human brain where APCs persist. Preliminaryobservations during physiological aging of the human brain havedemonstrated a marked regional and individual variation in thedistribution of the APC in adult human brain. In particular, Pax2⁺ GFAP⁻APCs and Pax2⁺ GFAP⁺ astroctyes have been identified in thesub-ventricular zone of the lateral ventricle of the adult brain. Incontrast, no Pax2⁺ GFAP⁻ APCs have been identified in thesub-ventricular zone of the third ventricle where GFAP⁺ Pax2⁻ adult/agedastrocytes have been identified.

EXAMPLE 20 Isolation and Purification of APCs from Human Fetal Brain andRetina and Determination of Culture Conditions that Support theExpansion and Controlled Differentiation of these Cells In Vitro

Human brain and retinal tissue are isolated from foetuses at the stageof development at which the number of these cells is maximal asdetermined by studies of intact fetal brain and retina.

EXAMPLE 21 Analysis of the Pattern of Gene Expression and theDevelopmental Potential In Vitro of Human APCs

The pattern of gene expression is examined in human APCs with the use ofDNA microarray analysis. The developmental potential of these cells isdetermined by clonal analysis in vitro.

EXAMPLE 22 Determination of the Ability of Rat APCs to Facilitate theMigration, Integration and Differentiation of Neural Precursor Cellswhen Co-Transplanted with these Latter Cells in Various ExperimentalModels of Neurodegenerative Conditions in which Astrocytes Play a Rolyin Pathophysiology

As groundwork for clinical trials of co-transplantation of APCs and stemcells and/or neural precursor cells, studies are undertaken aimed atcharacterizing the effects of implantation of rat APCs and stem cellsand/or neural precursor cells in various experimental models of CNStrauma and disease.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A method for generating a substantially homogeneous population oflineage-specific cells from tissue of the central nervous system (CNS)of mammalian animals, said method comprising subjecting said CNS tissueto tissue disruptive means to provide a mixed population of cellscomprising the lineage-specific cells to be isolated, subjecting themixed population to cell separation discrimination means to generate asubstantially homogeneous population of lineage-specific cells.
 2. Themethod of claim 1 wherein the lineage-specific cells are astrocyteprecursor cells (APCs).
 3. The method of claim 1 wherein thelineage-specific cells are immature perinatal astrocytes (IPAs).
 4. Themethod of claim 1 wherein the lineage-specific cells are matureperinatal astrocytes (MPAs).
 5. The method of claim 1 wherein thelineage-specific cells are adult or aged astrocytes.
 6. The method ofclaim 1 wherein the mammalian animal is at a prenatal stage.
 7. Themethod of claim 1 wherein the mammalian animal is at a postnatal stage.8. The method of claim 7 wherein the postnatal animal is an adult. 9.The method of claim 1 wherein the separation discrimination means isbased on a different range of cell markers present at differentdevelopmental stages of the lineage-specific cells.
 10. The method ofclaim 9 wherein the separation discrimination means is based on positiveor negative selection of cell surface markers.
 11. The method of claim10 wherein the neuronal cells are removed by negative selection withN-CAM.
 12. The method of claim 10 or 11 wherein glial cells are selectedpositively using one or more of A2B5, GD3, 3CB2, FGR3, PDGFRα or acombination thereof.
 13. The method of claim 10 or 11 or 12 whereinoligodendrocytes are removed by negative selection with one or more ofGal-C, 01, 04, anti-Mog or NG2 or a combination thereof.
 14. The methodof claim 9 wherein the cells are sorted by recognition of cell markersby immunological reagents.
 15. The method of claim 10 wherein theimmunoglobulin reagents are antibodies.
 16. The method of claim 2wherein the cells isolated are immunohistochemically Pax2⁺ GFAP⁻. 17.The method of claim 2 wherein the cells isolated areimmunohistochemically Pax2⁺ GFAP⁺.
 18. The method of any one of claims 1to 17 wherein the mammalian animal is a human.
 19. The method of any oneof claims 1 to 17 wherein the mammalian animal is a livestock animal,laboratory test animal or a companion animal.
 20. A substantiallyhomogeneous population of mammalian lineage-specific cells from the CNS,said cells made by the method comprising subjecting said CNS tissue totissue disruptive means to provide a mixed population of cellscomprising the lineage-specific cells to be isolated, subjecting themixed population to cell separation discrimination means to generate asubstantially homogeneous population of lineage-specific cells.
 21. Thepopulation of mammalian cells of claim 20 wherein the lineage-specificcells are astrocyte precursor cells (APCs).
 22. The method of claim 20wherein the lineage-specific cells are immature perinatal astrocytes(IPAs).
 23. The method of claim 20 wherein the lineage-specific cellsare mature perinatal astrocytes (MPAs).
 24. The method of claim 20wherein the lineage-specific cells are adult or agedd astrocytes. 25.The method of claim 20 wherein the mammalian animal is at a prenatalstage.
 26. The method of claim 20 wherein the mammalian animal is at apostnatal stage.
 27. The method of claim 26 wherein the postnatal animalis an adult or aged astrocyte.
 28. The method of claim 20 wherein theseparation discrimination means is based on a different range of cellmarkers present at different developmental stages of thelineage-specific cells.
 29. The method of claim 28 wherein theseparation discrimination means is based on positive or negativeselection of cell surface markers.
 30. The method of claim 29 whereinthe neuronal cells are removed by negative selection with N-CAM.
 31. Themethod of claim 28 or 29 or 30 wherein glial cells are selectedpositively using one or more of A2B5, GD3, 3CB2, FGR3, PDGFRα or acombination thereof.
 32. The method of claim 28 or 29 or 30 or 31wherein oligodendrocytes are removed by negative selection with one ormore of Gal-C, 01, 04, anti-Mog or NG2 or a combination thereof.
 33. Themethod of claim 28 wherein the cells are sorted by recognition of cellmarkers by immunological reagents.
 34. The method of claim 33 whereinthe immunoglobulin reagents are antibodies.
 35. The method of claim 20wherein APCs are isolated by the immunological separation of apopulation of Pax2⁺ cells followed by removal of GFAP⁺ cells form thePax2⁺ population.
 36. The method of claim 20 wherein IPAs are isolatedby the immunological separation of a population of vimentin⁺ cells andthen isolating GFAP⁺ cells from said vimentin⁺ population.
 37. Themethod of any one of claims 20 to 36 wherein the mammalian animal is ahuman.
 38. The method of any one of claims 20 to 36 wherein themammalian animal is a livestock animal, laboratory test animal or acompanion animal.
 39. A substantially homogeneous population of APCs orIPAs from the CNS such as brain including retina tissue prepared by themethod comprising subjecting said CNS tissue or part thereof to tissuedisruptive means and contacting said immobilized tissue with interactivemolecules to a cell marker selectively present or absent on or in saidAPCs or IPAs to generate a population comprising at least APCs and/orEPAs and then contacting the isolated cells with an interactive moleculeto at least one other cell marker specific for either said APCs or IPAsor specific for a cell marker absent from either APCs or IPAs toselectively remove or retain the desired cell type.
 40. A method of cellreplacement therapy in a mammalian animal, said method comprisinggenerating a substantially homogeneous population of lineage-specificcells and introducing same into an organ or tissue requiring cells to bereplaced or to another location from where the cells can migrate to anorgan or tissue requiring cells wherein the introduced cells are subjectto expansion or proliferation in vitro and/or in vivo by one or moregrowth factors.
 41. The method of claim 40 wherein the lineage-specificcells are astrocyte precursor cells (APCs).
 42. The method of claim 40wherein the lineage-specific cells are immature perinatal astrocytes(IPAs).
 43. The method of claim 40 wherein the lineage-specific cellsare mature perinatal astrocytes (MPAs).
 44. The method of claim 40wherein the lineage-specific cells are adult astrocytes.
 45. The methodof claim 40 wherein the mammalian animal is at a prenatal stage.
 46. Themethod of claim 40 wherein the mammalian animal is at a postnatal stage.47. The method of claim 46 wherein the postnatal animal is an adult. 48.The method of claim 40 wherein the separation discrimination means isbased on a different range of cell markers present at differentdevelopmental stages of the lineage-specific cells.
 49. The method ofclaim 48 wherein the separation discrimination means is based onpositive or negative selection of cell surface markers.
 50. The methodof claim 48 or 49 wherein the neuronal cells are removed by negativeselection with N-CAM.
 51. The method of claim 48 or 49 or 50 whereinglial cells are selected positively using one or more of A2B5, GD3,3CB2, FGFR3, PDGFRα or a combination thereof.
 52. The method of claim 48or 49 or 50 or 51 wherein oligodendrocytes are removed by negativeselection with one or more of Gal-C, Glc, 01, 04, anti-Mog or NG2 or acombination thereof.
 53. The method of claim 48 wherein the cells aresorted by recognition of cell markers by immunological reagents.
 54. Themethod of claim 53 wherein the immunoglobulin reagents are antibodies.55. The method of claim 40 wherein the cells isolated areimmunohistochemically Pax2⁺ GFAP⁻.
 56. The method of claim 40 whereinthe cells isolated are immunohistochemically Pax2⁺ GFAP⁺.
 57. The methodof claim 40 wherein IPAs are isolated by the immunological separation ofa population of vimentin⁺ cells and then isolating GFAP⁺ cells from saidvimentin⁺ population.
 58. The method of any one of claims 48 to 57wherein the mammalian animal is a human.
 59. The method of any one ofclaims 40 to 58 wherein the mammalian animal is a livestock animal,laboratory test animal or a companion animal.
 60. The method of claim 40wherein the therapy is for a degenerative disorder.
 61. The method ofclaim 60 wherein the degenerative disorder is Alzheimer's disease,Huntington's disease, HIV-associated dementia (HIV-D), a chronicneurological disorder, Parkinson's disease, epilepsy, stroke oralcoholism.
 62. The method of claim 60 wherein the degenerative disorderis hypoxia or a spinal chord injury.
 63. The method of claim 40 whereinthe therapy is an acute brain injury or CNS dysfunction.
 64. The methodof any one of claims 40 to 63 wherein the lineage-specific cells areco-introduced with neural stem cells or neuronal cells.
 65. The methodof claims 40 to 63 wherein the lineage specific cells are from the samesubject being treated.
 66. The method of any one of claims 40 to 63wherein the lineage-specific cells are from a different subject beingtested.
 67. A method for assessing the level of healthy tissue in a CNSbiopsy such as a brain biopsy in an adult subject said method comprisingdetermining in said biopsy presence of Pax2⁺ astrocyte cells wherein thepresence of said Pax2⁺ cells is indicative of a reversion in thematuration of said astrocytes.
 68. A composition of astrocyte precursorcells such as APCs or IPAs in substantially homogeneous form, saidcomposition optionally further comprising one or morepharmaceutically-acceptable carriers and/or diluents.
 69. Conditionedmedium from the in vitro culture of astrocyte precursor cells such asAPCs or IPAs wherein said conditioned medium comprises one or moregrowth factors or autocrine factors.
 70. A growth or autocrine factorobtainable from conditioned medium of an in vitro cell culture ofastrocyte precursors such as APCs or IPAs.
 71. A method for generating asubstantially homogenous population of mammalian cells of the astrocyticlineage, said method comprising isolating cell suspension from an adultbrain or embryonic brain and removing neural precursor cells by anegative selection using N-CAM or functional equivalent and thenselecting positively for glial cells using one or more of A2B5, GD3,3CB2, FGFR3, PDGFRα or a combination thereof or a functional equivalentthereof; culturing the resulting cells in a serum free medium togetherwith a growth factor and removing by negative selection oligodendrocytesusing markers Glc, Gal-C, 01, 04, anti-Mog or a combination thereof or afunctional equivalent thereof and then inducing differentiation alongthe maturation pathway by culturing cells in the presence of one or moreof CNTF, LIF, BMP such as BMP4, cAMP, TGFβ and/or EGF or functionalequivalents thereof.
 72. The method of claim 71 wherein the serum freemedium is DMEM/F-12.
 73. The method of claim 72 wherein the DMEM/F-12medium further comprises a growth factor selected from bFGF and chickembryo extract.
 74. Cells of the astrocytic lineage isolated by themethod of any one of claims 71 to 73.